Huang N N, Wang D J, Gonzalez F, Heppel L A
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.
J Cell Physiol. 1991 Mar;146(3):483-94. doi: 10.1002/jcp.1041460320.
We have previously shown that extracellular ATP acts as a mitogen via protein kinase C (PKC)-dependent and independent pathways (Wang, D., Huang, N., Gonzalez, F.A., and Heppel, L.A. Multiple signal transduction pathways lead to extracellular ATP-stimulated mitogenesis in mammalian cells. I. Involvement of protein kinase C-dependent and independent pathways in the mitogenic response of mammalian cells to extracellular ATP. J. Cell. Physiol., 1991). The present aim was to determine if metabolism of arachidonic acid, resulting in prostaglandin E2 (PGE2) synthesis and elevation of cAMP levels, plays a role in mitogenesis mediated by extracellular ATP. Addition of ATP caused a marked enhancement of cyclic AMP accumulation in 3T3, 3T6, and A431 cells. Aminophylline, an antagonist of the adenosine A2 receptor, had no effect on the accumulation of cyclic AMP elicited by ATP, while it inhibited the action of adenosine. The accumulation of cyclic AMP was concentration dependent, which corresponds to the stimulation of DNA synthesis by ATP. The maximal accumulation was achieved after 45 min, with an initial delay period of about 15 min. That the activation of arachidonic acid metabolism contributed to cyclic AMP accumulation and mitogenesis stimulated by ATP in 3T3, 3T6, and A431 cells was supported by the following observations: (a) extracellular ATP stimulated the release of [3H]arachidonic acid and PGE2 into the medium; (b) inhibition of arachidonic acid release by inhibitors of phospholipase A2 blocked PGE2 production, cyclic AMP accumulation, and DNA synthesis activated by ATP, and this inhibition could be reversed by adding exogenous arachidonic acid; (c) cyclooxygenase inhibitors, such as indomethacin and aspirin, diminished the release of PGE2 and blocked cyclic AMP accumulation as well as [3H]thymidine incorporation in response to ATP; (d) PGE2 was able to restore [3H]thymidine incorporation when added together with ATP in the presence of cyclooxygenase inhibitors; (e) pertussis toxin inhibited ATP-stimulated DNA synthesis in a time- and dose-dependent fashion as well as arachidonic acid release and PGE2 formation. Other evidence for involvement of a pertussis toxin-sensitive G protein(s) in ATP-stimulated DNA synthesis as well as in arachidonic acid release is presented. In A431 cells, the enhancement of arachidonic acid and cyclic AMP accumulation by ATP was partially blocked by PKC down-regulation, implying that the activation of PKC may represent an additional pathway in ATP-stimulated metabolism of arachidonic acid. In all of these studies, ADP and AMP-PNP, but not adenosine, were as active as ATP.(ABSTRACT TRUNCATED AT 400 WORDS)
我们之前已经表明，细胞外ATP通过蛋白激酶C（PKC）依赖和非依赖途径作为一种促有丝分裂原（Wang, D., Huang, N., Gonzalez, F.A., and Heppel, L.A. 多种信号转导途径导致哺乳动物细胞中细胞外ATP刺激的有丝分裂。I. 蛋白激酶C依赖和非依赖途径在哺乳动物细胞对细胞外ATP的有丝分裂反应中的作用。《细胞生理学杂志》，1991年）。目前的目的是确定花生四烯酸代谢产生前列腺素E2（PGE2）并提高cAMP水平是否在细胞外ATP介导的有丝分裂中起作用。添加ATP导致3T3、3T6和A431细胞中cAMP积累显著增强。氨茶碱是腺苷A2受体的拮抗剂，对ATP引起的cAMP积累没有影响，而它抑制腺苷的作用。cAMP的积累是浓度依赖性的，这与ATP对DNA合成的刺激相对应。在45分钟后达到最大积累，最初有约15分钟的延迟期。以下观察结果支持花生四烯酸代谢的激活促成了3T3、3T6和A431细胞中ATP刺激的cAMP积累和有丝分裂：（a）细胞外ATP刺激[3H]花生四烯酸和PGE2释放到培养基中；（b）磷脂酶A2抑制剂对花生四烯酸释放的抑制阻断了PGE2产生、cAMP积累以及ATP激活的DNA合成，并且通过添加外源性花生四烯酸可以逆转这种抑制；（c）环氧合酶抑制剂，如消炎痛和阿司匹林，减少了PGE2的释放并阻断了cAMP积累以及对ATP的[3H]胸苷掺入；（d）在环氧合酶抑制剂存在下，当与ATP一起添加时，PGE2能够恢复[3H]胸苷掺入；（e）百日咳毒素以时间和剂量依赖性方式抑制ATP刺激的DNA合成以及花生四烯酸释放和PGE2形成。还提供了其他证据表明百日咳毒素敏感的G蛋白参与ATP刺激的DNA合成以及花生四烯酸释放。在A431细胞中，PKC下调部分阻断了ATP对花生四烯酸和cAMP积累的增强作用，这意味着PKC的激活可能代表了ATP刺激的花生四烯酸代谢中的另一条途径。在所有这些研究中，ADP和AMP - PNP，但不是腺苷，与ATP一样具有活性。（摘要截断于400字）
